Development of novel isothermal/quantitative nucleic acid amplification method for ultra-high sensitive detection of biomolecules

Abstract

Isothermal nucleic acids amplification method has been a good alternative technique to polymerase chain reaction (PCR) for an achievement of point-of-care testing (POCT) or disease diagnosis. Particularly, due to the fact that it does not require any complicated equipment such as a thermal cycler and it exponentially amplifies target sequence in a short time, rapid, simple and sensitive target detection could be achieved. Additionally, as signaling methods such as fluorescent or colorimetric assay can be easily incorporated into isothermal amplification due to consistent temperature condition, it is applied to the sensitive and desirable detection of biologically important molecules. In this study, both the isothermal nucleic acids amplification methods for the genetic diagnosis including sexually transmitted diseases and for the detection of antigens were described. An iTPA (isothermal target and signaling probe amplification) method for the quantitative detection of nucleic acids, based on a combination of novel ICA (isothermal chain amplification) and FRET CPT (cycling probe technology), is described. In the new ICA method, two displacement events occur in the presence of four specially designed primers. This phenomenon leads to powerful amplification of target DNA. Since the amplification is initiated only after hybridization of the four primers, the ICA method leads to high specificity for the target sequence. As part of the new ICA method, iTPA is achieved by incorporating FRET CPT to generate multiple fluorescence signals from a single target molecule. By using the resulting dual target and signaling probe amplification system, even a single copy of a target gene can be successfully detected and quantified under isothermal conditions. A facile gold nanoparticle (AuNP)-mediated colorimetric method for real-time detection of target DNA in conjugation with our unique isothermal target and signaling probe amplification (iTPA) method, comprising novel ICA (i...